Proteomics Flashcards
What is proteomics?
Large-scale characterization of the entire protein complement of a cell-line, tissue or organism
Protein studies with analyses that have a genetic readout such as mRNA analysis, genomics, and the yeast two-hybrid analysis
What is the goal of proteomics?
To get a more global and integrated view of biology by studying all the proteins of a cell rather than each one individually; and create a complete 3-D map of the cell indicating where proteins are located
Why proteomics?
- one gene, multiple products
- Genome annotation
- Protein expression/function studies
- Protein-protein interactions
Describe the origin of proteomics
2D-E in 1975 by Proteomics pioneers such as:
- Patrick O’Farrell-high resolution 2D-E of Proteins (ELR1)
- Leigh Anderson proteomics studies of blood proteins and leukocytes
1st computerized 2-D gel image analysis platform was developed to quantitate changes in 2D gel protein spot levels
- Changes in protein abundance between samples could be quantitated
- frustration grew because of lack of tools to identify proteins of interest
2D gel reproducibility hindered the expansion of the technique until the introduction of immobilized pH gradients (IPGs) in 1982
Much improved 2nd generation IPGs in the late 80s, coincided with development of mass spectrometry ionization techniques for peptides, allowing protein identification and characterization on a large scale.
Mid 90s mass spectrometry became a mainstream technique for protein identification, mostly replacing Edman sequencing
Briefly describe proteomics technology
A typical proteomics experiment (e.g. protein expression profiling) can be broken down into the following steps:
- (I) the separation and isolation of proteins from a cell line, tissue, or organism
- (II) the acquisition of protein structural information for the purposes of protein identification and characterization
- (III) database utilization and functional validation
What methods can be used for separation and isolation of proteins?
- 1D gel electrophoresis (1D- E)
- 2D gel electrophoresis (2D-E)
- protein digestion
- purification
- isotope-coded affinity tags (ICAT)
What can western blot analysis be used for ?
To detect and quantify the amount of a particular protein:
- Proteins are resolved by size using SDS-PAGE gel electrophoresis
Following electrophoresis, most proteins within the protein sample can be visualized by staining the SDS-PAGEgel with Coomassie blue
-At this point, identification of proteins can only be approximated based on size
How can antibodies be used to detect and identify the presence of a specific protein?
Prior to using the antibody, the proteins resolved on the SDS-PAGE HDL must be transferred to a solid support.
-Solid support is typically nitrocellulose or nylon membrane
Transfer of protein utilizes an apparatus called an electroblotter
The resulting membrane containing the transferred proteins is called a Western blot
The proteins would have been coated with negative charges by the SDS. Therefore upon the application of electric current, the proteins will be repelled by the negative electrode and attracted by the positive electrode. This will result in migration of the proteins towards the positive electrode. The negative electrode is the cathode and the positive electrode is the anode
Summarize how western blotting can be used for protein detection
- Blocking blot is incubated with 5% skim milk to coat blot in non-specific protein.-attaches to the membrane in all places where the target proteins have not attached
- Blot is incubated with primary antibody
- Blot is incubated with a secondary antibody that cross reacts with the primary antibody. An enzyme is conjugated to the secondary antibody
- Visualization usually involves reaction of the enzyme with a chemiluminescent substrate
Contrast Coomassie blue staining and western blot
Coomassie blue staining-allows you to visualize several hundred proteins (many proteins are the same size and therefore can not be resolved separately )
Western blot- allows you to identify a single specific protein
Describe 2-D gel electrophoresis
Two dimensional gel electrophoresis is a valuable technique for separating proteins (in a protein extract from cells or tissues) which contain a complex mixture of proteins with varying biochemical properties
- In 2-D-E, proteins are separated in two sequential steps:
- first by their charge (1st dimension); and
- then by their mass (2nd dimension)
Describe how to conduct 2D-E experiment
1st dimension: load protein sample onto an isoelectric focusing tube gel. Electrophoresis separates proteins according to their isoelectric point, where their net charge is zero compared to the pH of the gel
2nd dimension: rotate tube gel 90 degreees and place onto an SDS-polyacrylamide gel (SDS-PAGE). Electrophoresis separates proteins according to mass (molecular weight in kilo daltons, kDa)
Stained gel shows proteins as a series of spots separated by isoelectric point and molecular mass
Describe the typical proteomics experiment
Expose cells to different conditions (like growth conditions, drugs or hormones)
- Extract proteins from the cells and separate by 2D-E
- Compare spots for evidence of differential expression
- Excise and digest spots of interest
- Analyze peptide fragments by Mass spectrometry to identify proteins
What is differential gel electrophoresis (DIGE)?
Proteins from different sample can be fluorescently labeled and used for 2D electrophoresis
Can allow comparison between protein samples
2D gels can be combined with western blotting to…
Identify specific proteins
Briefly describe protein digestion
It is difficult to obtain mass measurement of very large proteins
- Current Mass spectrometers are well suited to analyze peptide fragments
- Proteases would cleave proteins at specific amino acids residues to yield peptide fragments (ideally 6-20 amino acids long) that are most compatible with MS analysis
- Excise (cut) protein spots from gel and digest with a protease
Examples of proteases are:
- trypsin
- chymotrypsin
- Glu-C
- Lys-C
- Asp-N
List some peptide purification techniques
- Liquid chromatography
- Capillary ekectrophoresis
- Cation exchange chromatography
- Reverse-phase chromatography
What are Isotope-Coded Affinity Tags(ICAT)?
Labeling of protein samples
-from two different sources
- with two chemically identical reagents that differ only in mass as a result of isotope composition
Briefly describe acquisition of protein structure information
Edman sequencing
-micro sequencing to obtain N-terminal amino acid sequences
Mass spectrometry
- sample preparation
- sample ionization
- mass analysis
Describe Edman sequencing
Mixed peptide sequencing:
- complex protein mixture separated by SDS-PAGE(1DE or 2DE)
- proteins transferred to an inert membrane by electroblotting
- proteins of interest visualized on the membrane surface, excised
- fragment chemically at methionine (by cyanogen bromide-CMBr) or tryptophan (by skatole) to give 3-5 peptide fragments, consistent with the frequency of occurrence of methionine and tryptophan in most proteins
- membrane piece placed directly into an automated Edman sequencer without further manipulation
- 6 -12 automated Edmancycles carried out in 4-8 hours and mixed-sequence data fed into the FASTF or TFASTF algorithms, which short and match the data against protein (FASTF) and DNA(TFASTF) databases to unambiguously identify the protein
What is mass spectrometry?
Introduce sample (e.g. frommHPLC machine) to the instrument (Mass spectrometer)
Generate ions in the gas phase
Separate ions on the basis of differences in mass to charge (m/z) with analyzer
Detect ions and identify prote8ns via database
What are the methods of functional validation of proteins?
- Protein function microarrays
- Phage display approach
- yeast two-hybrid system
Describe protein function microarrays
Used to screen fir various types of protein interactions including
Protein-protein Protein-lipid Protein-DNA Protein-drug Protein peptide interactions
To identify enzyme substrates
To profile immune responses
Purified full length proteins or protein domains spotted onto nickel-coated microscope slides
Labeled probes
- nucleic acid
- proteins
- lipids-small molecules
What is the yeast two hybrid system?
Screening fir protein-protein interactions
- proteins called activators control gene expression in euksryotes
- An activator has to bind to DNA sequence upstream of a gene to stimulate RNA polymerase to copy the gene into RNA
Interaction between the human proteins, downstream reporter gene is expressed
Or no interaction between human proteins, no gene expression
What are the challenges of proteomics?
Genome- static, amplification possible , homogeneous molecules, no variability of the amount
Proteome- dynamic, no amplification possible, heterogenous molecules, large variability of the amount
